Project Summary Hsp70 molecular chaperones are central players in protein homeostasis and quality control. They mediate a diverse set of cellular functions using a deceptively simple allosteric mechanism. In their ATP-bound states, they bind substrates with rapid on/off rates and relatively low affinity, and in their ADP-bound states, substrates bind more tightly with slow association/dissociation. Hsp70s bind short sequences within unfolded regions of their substrates, preferring hydrophobic residues with flanking positively charged residues. Their transition between a high substrate affinity, ADP- bound state, and a low substrate affinity, ATP-bound state, involves major conformational rearrangement of both the N-terminal nucleotide-binding domain, and the C-terminal substrate- binding domain. While recent research has shed light on this allosteric conformational change, many questions remain and are the focus of the proposed research. What are the features of their substrate-binding sites that enable binding to many but not all sequences (they are “selectively promiscuous”)? Hsp70s work with partner co-chaperones, the J-proteins that help with cellular localization and delivery of specific substrates, and the nucleotide-exchange factors that facilitate replacement of ADP by ATP in the allosteric cycle. What are the structural origins of these partnerships, and how do they affect substrate binding and release? Preliminary results and literature observations suggest that Hsp70s can bind nearly isoenergetically to their extended polypeptide substrates in either an N- to C-orientation or a C- to N-orientation. This provocative bimodal substrate binding may have functional implications. The proposed work will examine the structural origins of this capability as well as the potential impact on the functions of the chaperone. Past work shows that the allosteric properties of Hsp70 are tunable by amino acid substitutions and by post-translational modifications. The proposed work will delve into the structural origins of this tuning, and the consequences of changes in the allosteric energy landscape for specific Hsp70 functions. Past work leaned heavily on peptide models to understand how Hsp70s bind their substrates. We will characterize the binding of Hsp70s to protein substrates to learn how the affinity for short sequence motifs translates into a hierarchy of site binding: are flanking sequences involved in selection of binding sites? How pivotal is accessibility? Lastly, the role of the Hsc70 chaperone in preparing SNAP-25 to participate in pre- synaptic vesicle docking and fusion in neurons will be studied. Methods that will be deployed in the proposed work include biochemical assays, nuclear magnetic resonance, fluorescence, mass spectrometry, and computational modeling.